Multicenter evaluation of two flow cytometric methods for counting low levels of white blood cells

BACKGROUND: Flow cytometric methods can be used to count residual white blood cells (WBCs) in WBC-reduced blood products, which should contain fewer than 1 x 10(6) WBCs per unit (approximately 3.3 WBCs/ microL). In this study two flow cytometric methods for counting WBCs under routine conditions in nine laboratories were evaluated. STUDY DESIGN AND METHODS: Panels of red blood cells (RBCs), platelets (PLTs), and plasma were prepared containing 33.3, 10.0, 3.3, 1.0, and 0.3 WBCs per microL and counted with flow cytometric methods (either LeucoCOUNT, BD Biosciences, four laboratories; or LeukoSure, Beckman Coulter, five laboratories). Requirements were that at the level of 3.3 WBCs per microL, coefficient of variation was < or =20 percent and accuracy was > or =80 percent. Routine flow cytometric quality control (QC) data of WBC-reduced blood products from two laboratories were analyzed. RESULTS: At the level of 3.3 WBCs per microL, none of the laboratories met the requirements for all three blood products. The LeucoCOUNT method met requirements at more laboratories than the LeukoSure method for RBCs and PLTs, but the opposite was true for plasma. Routine QC data showed that >99 percent of the flow cytometric measurements for WBC-reduced products was below the 95 percent prediction interval at 3.3 WBCs per microL. CONCLUSION: None of the laboratories met the requirements for accuracy and precision for all three blood products. Nevertheless, routine results showed that in >99 percent of the products, WBC counts were below guideline limits. Therefore, both flow cytometric methods are suitable for QC with pass-fail criterion.     

Differences in Residual White Blood Cell Subset Counts in Buffy Coat-Depleted Red Cell Concentrates Prepared with Bottom and Top or Quadruple-Bag Systems

Background: For preparation of buffy coat-depleted red cell concentrates (RCCs) in additive solution whole blood is currently collected in The Netherlands both in quadruple-bag and bottom and top bag systems. By using the quadruple-bag system both plasma and buffy coat cells are transferred into integrated satellite bags while the red cells remain in the collection bag. When bottom and top bags are used, the buffy coat remains in the collection bag while both red cells and plasma are transferred into satellite bags. The difference in processing prompted us to perform quantitative analysis of residual WBC subsets in buffy coat-depleted RCCs. Differences in removal of specific cells with the buffy coat could improve the outcome of additional filtration procedures aiming at complete removal of specific WBC subsets. Study Design and Methods: The buffy coat was removed in semiautomated procedures (Optipress I; Compomat G4) from units of whole blood collected in both bag systems. Paired samples were taken before and after removal of the buffy coat for counting and analyzing WBC subsets by flow cytometry using subset specific monoclonal antibodies. Results: All RCCs met the criteria from the guidelines of the Council of Europe. The percentage of residual total WBCs was lower (p < 0.001) in RCCs processed in bottom and top bag systems (26% Compomat and 18% Optipress) as compared to RCCs processed in quadruple-bag systems (43% compomat). WBC subset analysis in RCCs processed in quadruple-bag systems showed approximately 25% of residual T cells, B cells and monocytes and 60% of residual granulocytes. In contrast, WBC subset analysis in RCCs processed in bottom and top bag systems showed approximately 2% residual T cells, B cells, and monocytes and 35% residual granulocytes; in about 45% of units, lymphocytes and monocytes were even below the detection limit of flow cytometry analysis. Conclusion: Buffy coat-depleted RCCs are currently processed in bottom and top bag or quadruple-bag systems, the former being superior to the latter due to selective depletion of lymphocytes and monocytes by 98% (2 logs). The bottom and top procedure is an evident contribution to leukodepletion in blood transfusion, both with and without additional filtration.     

Platelets Stored in a New-Generation Container

Background and Objectives : In order to preserve platelet concentrates (PCs) with high yields, a new polyolefin platelet storage container (PL 2410, 1.3L, Baxter, La Châtre, France) with increased gas permeability in combination with a larger surface area has been developed. The storage capacity was studied with platelets in plasma and platelets additive solution. Materials and Methods : Platelet concentrate pools (PCs) of different yields suspended in either plasma (PCs-PL; n = 30) or PAS II (PCs-PAS; n = 37) were prepared. For preparation of PCs with a low, intermediate and high number of platelets 3, 5 and 6 buffy coat (BCs) were pooled with different volumes of plasma and 5 and 6 BCs were pooled with different volumes of PAS II, in order to obtain PCs of equal volumes comparable with routine conditions. All PCs were stored on a flatbed shaker at 22±2°C and evaluated on days 1, 2, 5 and 7 by determining platelet and white cell counts, pH (37°C), pO₂, pCO₂ and swirling score. Results : Platelet yields ranged from 1.5 up to 5.5 × 10¹¹ platelets/U. On day 7 all PCs-PL (n = 4) with platelet yields above 4.5×10¹¹ had a pH value below 6.8 (range 5.91–6.79). While 7 of 8 PCs-PAS units with platelet yields above 4.0×10¹¹/U showed a pH value below 6.8 (range 6.31–6.70). Conclusion : During 7 days of storage in a new 1.3-liter platelet container, the pH was maintained above 6.8 in PCs in plasma with a yield between 1.5-and 4.5×10¹¹/U; when PAS II was used, the maximum platelet yield allowed for comparable pH maintenance was somewhat lower (4.0x10¹¹/U).     

Hemolysis of red blood cells during processing and storage

BACKGROUND: During processing and storage, red blood cells (RBCs) undergo changes and cell injury resulting in hemolysis. Mostly, the separation of whole blood in top‐and‐bottom quadruple bag systems with break openings takes less than 4 minutes. However, longer separation times are not uncommon. The aims were to investigate whether hemolysis is increased in RBCs with longer separation time (RBCs_{>6 min}) compared to regular RBCs (RBCs_reg), to measure hemolysis increase during storage and to study whether frequency of hemolytic donations is donor dependent. STUDY DESIGN AND METHODS: RBCs_{>6 min} (n = 172) and 172 matched controls were tested for hemolysis on Days 1 and 21 RBC units from each group were stored at 4 ± 2°C and tested again after 5 weeks. Donor dependency was retrospectively investigated for 100 hemolytic RBC units. RESULTS: RBCs_{>6 min} exhibited a higher mean hemolysis rate than RBCs_reg (0.058% vs. 0.033%). Four RBC units were hemolytic (>0.8%), all RBCs_{>6 min} (2.36%). During storage, hemolysis in both groups increased with 0.24%. Hemolysis frequency did not seem to be donor dependent. CONCLUSIONS: Increased separation time is a useful screening tool for potentially increased hemolysis rate in RBCs. Hemolysis rate increased during storage equally in both groups. Hemolysis frequency appears donor independent.     

Allogeneic single-donor cryoseal produced from fresh-frozen quarantine apheresis plasma as alternative for multidonor or autologous fibrin sealants

BACKGROUND: Fibrin sealant is a human blood product consisting of two components: cryoprecipitate and thrombin. Commercial fibrin sealants are produced from multidonors, increasing the viral risk, and contain fibrinolytic inhibitors such as tranexamic acid or bovine aprotinin. Autologous fibrin sealants reduce the viral risk and are mostly produced during a surgical procedure or well in advance. Alternatively, the allogeneic single‐donor fibrin sealant cryoseal can be used. In this study cryoseal was characterized and the manufacturing consistency of the production process was investigated. STUDY DESIGN AND METHODS: Cryoseal was produced from plasma collected on apheresis machines using a commercial device. In a research setting the protein composition and recovery were determined. Also, the manufacturing consistency of the production process was tested in a research setting as well as in a routine setting. RESULTS: In the research setting all produced cryoseal met the quality control requirements of a clotting time of less than 10 seconds and the presence of Factor (F)XIII (qualitative). In the routine setting, one procedure per year did not meet these requirements. The protein composition showed the following mean ± standard deviation (%recovery) results: thrombin 25.7 ± 11.1 IU/mL, fibrinogen 19.9 ± 4.6 (15%) mg/mL, FVIII 15.6 ± 5.4 (44%) IU/mL, FXIII 2.7 ± 0.7 (6%) IU/mL, and plasminogen 1.8 ± 0.2 (4%) U/mL. In both research and routine settings the production process resulted in a consistent product. CONCLUSION: The cryoseal manufacturing process resulted in a consistent product, which meets the predetermined specifications. The single‐donor origin and the absence of fibrinolytic inhibitors make cryoseal a good alternative for multidonor and autologous fibrin sealants.     

WBC content of platelet concentrates prepared by the buffy coat method using different processing procedures and storage solutions

BACKGROUND: The number of WBCs in platelet concentrates (PCs) prepared by the buffy coat (BC) method with different storage solutions can result in low (5 x 10(6)/unit) WBC levels by the use of careful centrifugation techniques without filtration. At present, most blood banks use filtration steps to meet these requirements. The difference in processing methods and suspension solutions prompted the investigation of the influence of the various procedures on the WBC and platelet content of PCs. STUDY DESIGN AND METHODS: PCs from 5 BCs were harvested without or with inline filtration (AutoStop BC, Pall Corp.) in either plasma (PCs-plasma) or platelet additive solution (PCs-PAS-2). After preparation, samples were taken for counting WBCs and platelets and for analyzing WBC subsets by flow cytometry using specific MoAbs. The WBCs were concentrated before analysis of the WBC subsets. Results less than 2.5 cells per microL were considered below the limit of accuracy of the subset analysis. RESULTS: All filtered PCs met the AABB standard of 5 x 10(6) per unit and the European guidelines of 1 x 10(6) per unit. None of the nonfiltered PCs met the European guidelines, but all met the AABB guidelines. All filtered units gave residual WBC counts below the detection limit for subset analysis. Filtered PCs-plasma gave significantly higher platelet counts than filtered PCs-PAS-2 or nonfiltered PCs (p<0.01, ANOVA). CONCLUSION: Careful centrifugation of pooled BCs, with plasma or PAS-2, can result in PCs with low WBC contamination levels. However, filtered PCs are superior, because of better WBC removal and higher platelet counts.     

The effect of whole-blood storage time on the number of white cells and platelets in whole blood and in white cell-reduced red cells

BACKGROUND: Whole blood (WB) can be stored for some time before it is processed into components. After introduction of universal white cell (WBC) reduction, it was observed that longer WB storage was associated with more residual WBCs in the WBC-reduced red cells (RBCs). Also, weak propidium iodide (PI)-positive events were observed in the flow cytometric WBC counting method, presumably WBC fragments. The effect of storage time on the composition of WB and subsequently prepared WBC-reduced RBCs was studied. STUDY DESIGN AND METHODS: WB was collected in bottom-and-top collection systems with inline filters, obtained from Baxter, Fresenius, or MacoPharma. Units were stored at room temperature and separated into components in 4-hour intervals between 4 and 24 hours after collection. RBCs were WBC-reduced by inline filtration (approx. 50/group). RESULTS: Platelet (PLT) counts were lower in WB stored for 4 to 8 hours compared to 20 to 24 hours (mean +/- SD): 79 +/- 31 versus 102 +/- 30 for Baxter (p < 0.01); 91 +/- 31 versus 101 +/- 35 for Fresenius (not significant); and 73 +/- 47 versus 97 +/- 31 (all x 10(9) per unit) for MacoPharma (p < 0.01), respectively. The median residual WBC counts in WBC-reduced RBCs for WB stored for 4 to 8 and 20 to 24 hours were 0.03 versus 0.17 for Baxter (p < 0.001), 0.00 versus 0.06 for Fresenius (p < 0.001), and 0.13 versus 0.26 (all x 10(6) per unit) for MacoPharma (not significant), respectively. All WBC-reduced RBCs contained fewer than 5 x 10(6) WBCs per unit. A longer storage time of WB was associated with more weak PI-positive events, irrespective of the filter. CONCLUSION: Longer storage of WB before processing results in counting higher numbers of PLTs in WB, higher numbers of WBCs in WBC-reduced RBCs, and more weak PI-positive events.     

Evaluation of a strategy to limit blood donor exposure in high risk premature newborns based on clinical estimation of transfusion need

OBJECTIVES: Reservation of dedicated series of pedipacks, consisting of 3 to 4 units of 70 ml filtered red cell concentrate in additive solution SAGM from 1 donor, may reduce donor exposure. In this prospective efficacy study the benefits, release and expiration of pedipacks (PP) assigned to preterm infants requiring neonatal intensive care are analyzed. METHODS: On the basis of clinical assessment of the need for multiple transfusions, 96 preterm neonates (gestational age < 32 wks and/or birth weight < 1500 g) were assigned to either the high risk group (HRG), who were to receive dedicated donor blood units, or the low risk group (LRG). Inclusion criteria for HRG were 1) estimated time of admission > 21 days and 2) expected need for multiple transfusions due to clinical cardiorespiratory instability, prolonged parental feeding or frequent blood sampling. To reduce wastage of donor blood, dedication of donor blood units was limited to 21 days. RESULTS: 50 series (192 PP) were assigned to 42 HRG infants. Two HRG infants received 3 series, 4 received 2 series and 36 received 1 series of PP. Mean transfusion rate was 3.1 PP in the HRG and 0.4 in the LRG. In the LRG 35 of 54 were not transfused, 19 received 1 to 2 PP. In both groups transfused newborns were exposed to 1.1 donors in average. In the HRG of 192 PP, 137 PP (71%) were used within 21 days, and another 30 (16%) before the expiration date < 35 days. Twenty five PP (13%) expired, mainly because of logistical problems in the introduction phase. CONCLUSION: Assignment of dedicated PP on the basis of clinical parameters at entry considerably reduces donor exposure in HRG. Wastage of dedicated blood transfusions was reduced by limitation of the dedicated period (21 days). In terms of efficacy, reservation and use of PP can be optimized by standardized administrative measures.